Juvenile zebrafish (Danio rerio) are able to recover from lordosis.

Haemal lordosis, a frequent skeletal deformity in teleost fish, has long been correlated with increased mechanical loads induced by swimming activity. In the present study, we examine whether juvenile zebrafish can recover from haemal lordosis and explore the musculoskeletal mechanisms involved. Juveniles were subjected to a swimming challenge test (SCT) that induced severe haemal lordosis in 49% of the animals and then immediately transferred them to 0.0 total body lengths (TL) per second of water velocity for a week. The recovery from lordosis was examined by means of whole mount staining, histology and gene expression analysis. Results demonstrate that 80% of the lordotic zebrafish are capable of internal and external recovery within a week after the SCT. Recovered individuals presented normal shape of the vertebral centra, maintaining though distorted internal tissue organization. Through the transcriptomic analysis of the affected haemal regions, several processes related to chromosome organization, DNA replication, circadian clock and transcription regulation were enriched within genes significantly regulated behind this musculoskeletal recovery procedure. Genes especially involved in adipogenesis, bone remodeling and muscular regeneration were regulated. A remodeling tissue-repair hypothesis behind haemal lordosis recovery is raised. Limitations and future possibilities for zebrafish as a model organism to clarify mechanically driven musculoskeletal changes are discussed.

Evaluation of the impact of polyethylene microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae.

Microplastics are present in marine habitats worldwide and may be ingested by low trophic organisms such as fish larvae, with uncertain physiological consequences. The present study aims at assessing the impact of polyethylene (PE 10-45 µM) microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Fish were fed an inert diet including 0, 10(4) and 10(5) fluorescent microbeads per gram from 7 until 43 days post-hatching (dph). Microbeads were detected in the gastrointestinal tract in all fish fed diet incorporating PE. Our data revealed an efficient elimination of PE beads from the gut since no fluorescent was observed in the larvae after 48 h depuration. While the mortality rate increased significantly with the amount of microbeads scored per larvae at 14 and 20 dph, only ingestion of the highest concentration slightly impacted mortality rates. Larval growth and inflammatory response through Interleukine-1-beta (IL-1ß) gene expression were not found to be affected while cytochrome-P450-1A1 (cyp1a1) expression level was significantly positively correlated with the number of microbeads scored per larva at 20 dph. Overall, these results suggest that ingestion of PE microbeads had limited impact on sea bass larvae possibly due to their high potential of egestion.

Mechanism of action of ethylene sulfite and vinylene carbonate electrolyte additives in LiNi1/3Mn1/3Co1/3O2/graphite pouch cells: electrochemical, GC-MS and XPS analysis.

The role of ethylene sulfite used either alone or in combination with VC in LiNi1/3Mn1/3Co1/3O2 (NMC)/graphite pouch cells was studied by correlating data from differential capacity (dQ/dV) analysis, gas chromatography/mass spectroscopy (GC-MS), theoretical calculations, ultrahigh precision coulometry, storage experiments and X-ray photoelectron spectroscopy. For cells containing VC alone, the electrochemical performance and gas production were greatly improved, compared to cells without VC, due to the formation of more stable and protective SEI films at both electrode surfaces by a polymer of VC. For cells with ES alone, a vigorous reactivity was observed due to preferential reduction that also generated large amounts of gas during formation. The dramatic decrease in electrochemical performance as well as the continuous production of gas during cycling in cells with ES was explained by the formation of a very thin and ineffective SEI film at the NMC surface. The suppression of the vigorous reaction of ES in cells with both ES and VC occurred because the solvation energy of Li(+) by VC is smaller than that of EC so VC is reduced first during formation. During charge-discharge cycling, a slow consumption of ES occurred and different sulfur species were observed on the electrodes when VC was combined with ES. SEI film formation processes and SEI composition were therefore dominated by VC and the electrochemical performance of cells with both VC and ES were similar compared to those of cells with VC alone.

Redirected charge transport arising from diazonium grafting of carbon coated LiFePO4.

The morphological and the electrical properties of carbon coated LiFePO4 (LFPC) active material functionalized by 4-ethynylbenzene tetrafluoroboratediazonium salt were investigated. For this purpose, FTIR, Raman, XPS, High Resolution Transmission Electron Microscopy (HRTEM) and Broadband Dielectric Spectroscopy (BDS) were considered. Electronic conductivities of LFPC samples at room temperature were found to decrease in a large frequency range upon simple immersion in polar solvents and to decrease further upon functionalization. Due to their high dipole moment, strongly physisorbed molecules detected by XPS likely add barriers to electron hopping. Significant alteration of the carbon coating conductivity was only observed, however, upon functionalization. This effect is most presumably associated with an increase in the sp(3) content determined by Raman spectroscopy, which is a strong indication of the formation of a covalent bond between the organic layer and the carbon coating. In this case, the electron flux appears to be redirected and relayed by short-range (intra chain) and long-range (inter chain) electron transport through molecular oligomers anchored at the LFPC surface. The latter are controlled by tunnelling and slightly activated hopping, which enable higher conductivity at low temperature (T < 250 K). Alteration of the electron transport within the carbon coating also allows detection of a relaxation phenomenon that corresponds to small polaron hopping in bulk LiFePO4. XPS and HRTEM images allow a clear correlation of these findings with the island type oligomeric structure of grafted molecules.

Do ice nucleating agents limit the supercooling ability of the land snail Cornu aspersum?

The supercooling ability of adults and eggs of the partially freezing tolerant land snail Cornu aspersum remains limited to high subzero temperatures (ca. -5 degree C) whatever the conditions, suggesting the presence of ice nucleating agents (INAs). In this study, we investigated the nucleation activity of the digestive tract of adult snails, eggs and their direct environment: food, faeces and soil. The mucous ribbon always present in the distal intestine of adults exhibited a heat-sensitive (i.e. organic) nucleation activity, close to that of the entire snails during dormant states (aestivation and hibernation). However, a microbial nature of these INAs could not be established in inactive snails. The food provided to active snails contained ice nucleating bacteria, which followed the digestive tract to be found in the intestine and in the faeces, but with a decreasing concentration along the transit. Eggshells also presented a heat-sensitive nucleation activity, which could be related to its structure. Moreover, eggs are laid directly in the soil which contained both organic and mineral INAs. This study is the first to demonstrate the implication of organic INAs in the cold hardiness of a terrestrial gastropod.

Shell shape of the land snail Cornu aspersum in North Africa: unexpected evidence of a phylogeographical splitting.

Anatomical and molecular characters used to differentiate populations of the land snail Cornu aspersum (Helix aspersa) exhibit, in the western Mediterranean, definite and concordant patterns of correlation with geography. Scenarios involving Pliocene geological changes and postglacial expansion during the Pleistocene were proposed in previous studies to account for the establishment of this geographical structure. In the present work, we have performed a spatial analysis of variation in shell morphometrics, after the partitioning of the overall variation into size and shape components by means of a principal component-based approach (Cadima and Jolliffe, 1996). In order to know if the same historical events have also structured shell variation, the analysis includes all the populations from North Africa which were investigated for anatomical and molecular surveys. Contrary to shell size, which shows a significant spatial heterogeneity essentially related to environmental pressures, variation in shell shape components splits the populations according to a geographical pattern reflective of hypotheses suggested for molecular markers and genital anatomy. This implies that the selective forces often invoked to explain spatial changes in shell shape are not the deciding factors in the present case. Moreover, within each of the two geographical clusters defined, Mantel correlograms show that the similarity between populations declines according to an isolation by distance model. Because of the different allometric relationships between shell size and genitalia measurements in Western and Eastern entities of North Africa, mechanical constraints, possibly leading to a precopulatory isolation in the contact zone, are involved.

Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata).

Local patterns of genetic variation were analysed in the land snail Helix aspersa for 32 populations sampled within a patchy agricultural landscape: the polders of the Bay of Mont-Saint-Michel (France). This investigation examined the allele frequencies at four enzymatic markers and five microsatellite loci through the genotyping of 580 individuals. A strongly significant population genetic substructuring (mean F(ST)=0.088, P<0.001) was found at the scale of the whole polders area (3050 ha) and both categories of markers displayed a similar magnitude of spatial genetic differentiation. We did not find any obvious effects of habitat fragmentation on the distribution of genetic variability. Despite the reality of habitat patchiness and environmental instability (related to farming practices), an isolation by distance process was clearly depicted, although selective pressures cannot be ruled out for one enzymatic locus. Overall, genetic drift, along with occasional long-distance episodes of gene flow, was presumably the most likely evolutionary force that shaped the observed pattern of genetic variation.

Spatial structure of shell polychromatism in populations of Cepaea nemoralis: new techniques for an old debate.

A conspicuous shell polychromatism is observed in colonies of Cepaea nemoralis from western France (Brittany). The present study is intended to search for a spatial structure of shell features at this scale and to infer evolutionary processes from the observed patterns. We used a database of morph frequencies (six composite phenotypes were retained) measured on 213 samples regularly distributed on the whole studied area. Data analysis was based on two distinct multivariate methods leading to the following steps: (i) to search for a structure without reference to environmental conditions with a method (global principal components analysis: GPCA) which takes into account the spatial information by means of a neighbouring relationship between sampling points (Delaunay triangulation); (ii) to test the structuring power of environmental conditions by means of two explanatory factors (distance from the sea, altitude) involved in a redundancy analysis (RDA); (iii) to search for a spatial structure using residuals of the previous analysis, ie, after removing effects of environmental conditions. Global covariance accounted for 26.4% of the total variance, leading to a highly significant autocorrelation for each phenotype (step 1). Geographical mapping of factorial scores resulting from global analysis showed a well structured littoral zone and a strong southern-northern inland differentiation. Sixteen percent of the total variance was expressed in RDA but all morphs were not equally concerned. After removing environmental effects, a significant spatial structure still remains but was essentially caused by random processes. We argue for the importance of these last phenomena.

Spatial analysis of allozyme and microsatellite DNA polymorphisms in the land snail Helix aspersa (Gastropoda: Helicidae).

The genetic structure of the land snail Helix aspersa was investigated for 21 populations collected along a road located in the polders of the Bay of Mont-Saint-Michel (Brittany, France), following a sampling scheme the area of which did not exceed 900 m in length. A total of 369 individuals were genotyped for five enzymatic markers and seven microsatellite loci. We used sequential hierarchical F-statistics at different spatial scales and spatial autocorrelation statistics to explore recent historical patterns involved in the observed genetic distribution. Whatever the statistics used, congruent levels of spatial genetic substructuring across loci were demonstrated, excepted for one allozyme locus. Overall spatial genetic arrangement matched in a substantial fashion theoretical predictions based on the limited dispersal power of land snails. Positive autocorrelation over short-distance classes may result from the development of genetically distinct patches of individuals organized in family-structured colonies. Therefore, spatial signatures of average I correlograms can be viewed as the expression of a stepping-stone model of population structure, sometimes involving external migrational events. Overall, the revealed pattern of population subdivision on a microgeographical scale was suggestive of a neighbourhood structure. Finally, microsatellite loci are especially suitable for the detection of small genetic clustering, and combining different classes of markers offers the potential to gain further insight into the description of spatial genetic variability over short temporal and geographical scales.

Evolutionary history of the land snail Helix aspersa in the Western Mediterranean: preliminary results inferred from mitochondrial DNA sequences.

Intraspecific phylogeographic methods provide a means of examining the history of genetic exchange among populations. As part of a study of the history of Helix aspersa in the Western Mediterranean, we performed a phylogenetic analysis based on partial sequences of the mitochondrial large ribosomal subunit (16S) gene. Our samples include 31 H. a. aspersa populations from North Africa previously investigated for anatomical and biochemical characters. To clarify subspecific relationships, three individuals of the subspecies H. a. maxima were also studied. The molecular phylogeny inferred agrees largely with previous results, in splitting H. a. aspersa haplotypes into an eastern and a western group. H. a. maxima haplotypes form a third lineage arising before the H. a. aspersa groups. Divergence times estimated between the lineages suggest that dispersal during Pleistocene glaciation and vicariance events due to Pliocene geological changes in the western Mediterranean may both have played a significant part in the establishment of the present range of H. aspersa.

[Two years follow-up of a heroin users cohort treated with high dosage buprenorphine. Results of the SPESUB study (pharmacoepidemiologic follow-up of general practice Subutex)]. / Suivi à 2 ans d'une cohorte de patients sous buprénorphine haut dosage. Résultats de l'étude SPESUB (suivi pharmaco-épidémiologique du Subutex en médecine de ville).

BACKGROUND: Since February 1996, French GPs are allowed to prescribe high dosage buprenorphine for maintenance treatment of major opioid drug addiction. A prospective cohort of major opioid addicts was initiated in order to assess patient outcomes: follow-up, retention rate in treatment, drug use, intravenous injection and social situation evolution. METHODS: Each GP, known to be involved in drug user management, had to include the first 10 opioid drug addict patients to whom he prescribed high dosage buprenorphine, with a maximum inclusion period of 3 months. Patients were followed up for two years and a regular standardized information was collected (usual data on drug users and prescription modalities). RESULTS: Between May and July 1996, 919 patients (664 men and 255 women, mean age: 30 years) were included by 101 GPs. They had a long and serious history of drug addiction, important parallel consumption of cocaine, codeine and other illicit drugs and psychiatric problems (28% of definite problems and 45% of probable) and frequent hepatic conditions (hepatitis B: 23%, hepatitis C: 21%). Two years later, 55% of patients were still followed-up by the same GP and an additional 12% were followed by another GP or in a health care service (hospitalized or receiving methadone in a specialized centre). 13% were not followed, but GPs were able to describe their situation. 8% had been included by GPs who had dropped the study. Finally, 12% of patients were lost to follow-up. Among the 508 patients still followed-up by the same GP after 2 years, the substitution treatment rate was 84%. The dosage bracket had widened (inclusion: mean dosage=7.8 mg +/-4.5, minimum=0.8, maximum=28, median=8; after 2 years: mean=7.6 mg +/-5.4, minimum=0.4, maximum=28, median=8) and the duration of the prescription and dispensing had increased. Declaration of heroin intake in the previous month had fell from 40% to 11% and declaration of drug intake from 53% to 20%. Social situation had improved on average (housing conditions and work). There were 12 seroconversions for hepatitis B, 21 for hepatitis C and 4 for HIV. 14% of patients had declared intravenous injection of high dosage buprenorphine in the previous month. CONCLUSION: After two years of follow-up, 55% of patients were still followed-up by the same GP and an additional 12% was followed by another GP or in a health care service. Among patients still followed up by the same GP, a reduction of drug related harm (seroconversions for hepatitis B, hepatitis C and HIV) was observed.

Microspatial genetic structure in the land snail helix aspersa (Gastropoda: helicidae)

The microspatial genetic structure of allele frequencies at seven isozyme loci was examined for 15 populations of the land snail Helix aspersa sampled in a village from Brittany (north-western France). Spatial heterogeneity of allele frequencies was highly significant (P < 0.001). Fixation indices reflected nonrandom mating within neighbourhoods and a slight but consistent differentiation between colonies (FST=0.044; P < 0.01). Analyses of gene flow or genetic distances failed to reveal a significant relationship with geographical distance, probably because of the complexity of environmental heterogeneity. However, matrix comparisons between genetic distances and connectivity networks among adjacent colonies (Gabriel-connected graph) yielded a significant correlation in every case, indicating a 'step-by-step' relationship between neighbouring localities. Moreover, most of the allozymes were spatially structured and showed (i) a gradual isolation of colonies with increasing geographical distances, and (ii), for some correlogram profiles, a circular gradient illustrating a multidirectional colonization of the village. The probable existence of disperser individuals allowed us to suggest a metapopulation model which would explain the maintenance of such animals in fragmented habitats where anthropogenic disturbances and extinction/recolonization events are commonly observed.

Genetic distances and ordination: the land snail Helix aspersa in north Africa as a test case.

We examined the efficiencies of ordination methods in the treatment of gene frequency data at intraspecific level, using metric and nonmetric distance measures (Nei's and Rogers' genetic distances, chi 2 distance). We assessed initial processes responsible for the geographical distribution of the Mediterranean land snail Helix aspersa. Seventeen enzyme loci from 30 North African snail populations were considered in the present analysis. Five combinations of distance/multivariate analysis were compared: correspondence analysis (CA), nonmetric multidimensional scaling (NMDS) on Nei's, Rogers', and chi 2 distances, and principal coordinates analysis on Rogers' distances. Configuration of the objects resulting from ordination was projected onto three-dimensional graphics with the minimum spanning tree or the relative neighborhood graph superimposed. Pre- and postordination or clustering distance matrices were compared by means of correlation methods. As expected, all combinations led to a clear west versus east pattern of variation. However, the intraregional relationships and degree of connectivity between pairs of operational taxonomic units were not necessarily constant from one method to another. Ordination methods when applied with Nei's and Rogers' distances provided the best fit, with original distances (r = 0.98) compared with UPGMA clustering (r approximately 0.75). The Nei/NMDS combination seems to be a good compromise (distortion index dt = 10%) between Rogers/NMDS, which produces a more confusing pattern of differentiation (dt = 24%), and chi 2/CA, which tends to distort large distances (dt = 31%). NMDS obviously provides a powerful method to summarize relationships between populations, when neither hierarchical structure nor phylogenetic inference are required. These findings led the discussion on the good performance of NMDS, the appropriate distances to be used, and the potential application of this method to other types of allelic data (such as microsatellite loci) or data on nucleotide sequences of genes.

[Variability of reproduction examined in the laboratory between natural populations of Helix aspersa Müller from the Brittany region]. / Variabilité de la reproduction examinée au laboratoire entre populations naturelles d'Helix aspersa Müller de la région Bretagne.

Laboratory experiments were conducted to investigate the effects of geographical localization of natural populations and sampling dates on the reproduction of the garden snail Helix aspersa Müller. In Brittany, reproductive variability between populations shows the adaptive flexibility of characters describing reproductive activity and the relative stability of reproductive ability.